HATRIC-based identification of receptors for orphan ligands

Nadine Sobotzki,Sandra Goetze,Anika Koetemann,Yohei Yamauchi,Michael A Schafroth,Erick M Carreira,Florian Marty,Alina Rudnicka,Bernd Wollscheid

doi:10.1038/s41467-018-03936-z

Nadine Sobotzki, Sandra Goetze + Show 7 more

Open Access

https://doi.org/10.1038/s41467-018-03936-z

Copy DOI

Abstract

Cellular responses depend on the interactions of extracellular ligands, such as nutrients, growth factors, or drugs, with specific cell-surface receptors. The sensitivity of these interactions to non-physiological conditions, however, makes them challenging to study using in vitro assays. Here we present HATRIC-based ligand receptor capture (HATRIC-LRC), a chemoproteomic technology that successfully identifies target receptors for orphan ligands on living cells ranging from small molecules to intact viruses. HATRIC-LRC combines a click chemistry-based, protein-centric workflow with a water-soluble catalyst to capture ligand-receptor interactions at physiological pH from as few as 1 million cells. We show HATRIC-LRC utility for general antibody target validation within the native nanoscale organization of the surfaceome, as well as receptor identification for a small molecule ligand. HATRIC-LRC further enables the identification of complex extracellular interactomes, such as the host receptor panel for influenza A virus (IAV), the causative agent of the common flu.

Highlights

Cellular responses depend on the interactions of extracellular ligands, such as nutrients, growth factors, or drugs, with specific cell-surface receptors
Given the experimental setup, the candidates identified from HATRIC-Ligand-based receptor capture (LRC) experiments can generally result from the following four scenarios: (1) there is a direct interaction of the ligand with the target receptor; (2) the protein is in close proximity of the target receptor (“neighborhood protein”); (3) the protein is upregulated in response to treatment with the ligand and is overrepresented in the background binding of HATRIC or (4) the identified candidate is a false positive
A single HATRIC-LRC experiment does not allow us to delineate which type of interaction is taking place, but the validation experiments and the cited data clearly underline the relevance of the identified proteins

Summary

Introduction

Cellular responses depend on the interactions of extracellular ligands, such as nutrients, growth factors, or drugs, with specific cell-surface receptors. The sensitivity of these interactions to non-physiological conditions, makes them challenging to study using in vitro assays. Application of TRICEPS-LRC and ASB in different biological systems revealed the need to redesign the first-generation technologies: TRICEPS-LRC was intentionally designed to enable the identification of ligand-bound receptors solely based on formerly N-glycosylated peptides. We develop a new LRC technology with catalystenhanced cell surface labeling and protein-level affinity purification that enables the capture of ligand-receptor interactions at pH 7.4 from as few as 1 million cells

Methods

Results

Conclusion